Journal of Fungi Article Glycosylphosphatidylinositol Anchors from Galactomannan and GPI-Anchored Protein Are Synthesized by Distinct Pathways in Aspergillus fumigatus Jizhou Li 1, Isabelle Mouyna 1, Christine Henry 1, Frédérique Moyrand 2, Christian Malosse 3, Julia Chamot-Rooke 3, Guilhem Janbon 2, Jean-Paul Latgé 1 and Thierry Fontaine 1,* 1 Unité des Aspergillus, 25 rue du Docteur Roux, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France; [email protected] (J.L.); [email protected] (I.M.); [email protected] (C.H.); [email protected] (J.-P.L.) 2 Unité de Biologie des ARN des Pathogènes Fongiques, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France; [email protected] (F.M.); [email protected] (G.J.) 3 Unité de Spectrométrie de Masse pour la Biologie, Institut Pasteur, CNRS USR 2000, 28 rue du Docteur Roux, 75015 Paris, France; [email protected] (C.M.); [email protected] (J.C.-R.) * Correspondence: [email protected]; Tel.: +33-145-688-358 Received: 8 December 2017; Accepted: 19 January 2018; Published: 2 Febuary 2018 Abstract: Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1—coding for a phospholipase required for the first step of the GPI lipid remodeling—was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the Dper1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the Dper1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway. Keywords: glycosylphosphatidylinositol; Aspergillus fumigatus; PER1; cell wall; galactomannan 1. Introduction Glycosylphosphatidylinositols (GPI) are specific glycolipids that allow attachment of soluble proteins at the outlet layer of the plasma membrane. GPI-anchored proteins (GPI-AP) are ubiquitously found in eukaryotic organisms and are involved in several biological functions such as ligand recognition, enzymatic activities, cell–cell interaction, and host defense [1]. The sequence of GPI-APs is characterized by the presence of a hydrophobic peptide signal for GPI anchoring at the C-terminal. Using an in silico analysis, 115 GPI-APs have been predicted in Aspergillus fumigatus [2]. In the yeast model Saccharomyces cerevisiae, a number of GPI-APs are required for organization and remodeling of the cell wall, making the GPI biosynthesis an essential pathway to fungal life and morphogenesis. The fungal cell wall is mainly composed of polysaccharides, chitin, α and β-glucans, galactomannan, and galactosaminogalactan, organized in a three-dimensional network [3]. J. Fungi 2018, 4, 19; doi:10.3390/jof4010019 www.mdpi.com/journal/jof J. Fungi 2018, 4, x FOR PEER REVIEW 2 of 14 J.morphogenesis. Fungi 2018, 4, 19 The fungal cell wall is mainly composed of polysaccharides, chitin, α and β-glucans,2 of 14 galactomannan, and galactosaminogalactan, organized in a three-dimensional network [3]. In addition to GPI-anchored polysaccharide hydrolases such as chitinases and glucanases, three specific GPI- In addition to GPI-anchored polysaccharide hydrolases such as chitinases and glucanases, three specific anchored transglycosidase activities are required for the building of the cell wall skeleton: (i) Two types GPI-anchored transglycosidase activities are required for the building of the cell wall skeleton: (i) Two of β-1,3-glucan remodelase have been characterized first in A. fumigatus [4,5]: the β-1,3-glucan types of β-1,3-glucan remodelase have been characterized first in A. fumigatus [4,5]: the β-1,3-glucan branching enzyme and the GAS/GEL family members responsible for β-1,3-glucan chain elongation. branching enzyme and the GAS/GEL family members responsible for β-1,3-glucan chain elongation. These β-1,3-glucanosyltransferase activities are essential for the biosynthesis of the fungal cell wall [6– These β-1,3-glucanosyltransferase activities are essential for the biosynthesis of the fungal cell 8]; (ii) The Crh family (for congo red hypersensitive) contains putative transglucosidase activity wall [6–8]; (ii) The Crh family (for congo red hypersensitive) contains putative transglucosidase involved in the β-glucan-chitin reticulation [9]. Five orthologs have been identified in A. fumigatus, activity involved in the β-glucan-chitin reticulation [9]. Five orthologs have been identified in A. but their function remains unknown; (iii) The GPI-anchored Dfg (Defect in filamentous growth) fumigatus, but their function remains unknown; (iii) The GPI-anchored Dfg (Defect in filamentous family is composed of two proteins in yeast with redundant activities. Double knockout was growth) family is composed of two proteins in yeast with redundant activities. Double knockout was synthetically lethal [10]. In A. fumigatus, seven orthologs have been identified and are required for synthetically lethal [10]. In A. fumigatus, seven orthologs have been identified and are required for the the galactomannan cross-linking onto the cell wall β-1,3-glucan [11]. galactomannan cross-linking onto the cell wall β-1,3-glucan [11]. Fungal GPI-anchors are composed of a common glycan structure containing 4 mannose residues Fungal GPI-anchors are composed of a common glycan structure containing 4 mannose residues linked to a glucosamine. The anchored protein is linked to the third mannose residue of GPI through a linked to a glucosamine. The anchored protein is linked to the third mannose residue of GPI phosphoethanolamine bridge and one more mannose residue may be added to the common core [12,13] through a phosphoethanolamine bridge and one more mannose residue may be added to the common (Figure 1). The second particularity of fungal GPI is based on the lipid moiety that is an inositol- core [12,13] (Figure1). The second particularity of fungal GPI is based on the lipid moiety that is an phosphoceramide [13,14]. In addition to GPI-APs, A. fumigatus produces a lipogalactomannan (LGM) inositol-phosphoceramide [13,14]. In addition to GPI-APs, A. fumigatus produces a lipogalactomannan which is, to date, the sole fungal GPI-anchored polysaccharide [15]. The LGM is constituted by the (LGM) which is, to date, the sole fungal GPI-anchored polysaccharide [15]. The LGM is constituted by elongation of the mannan moiety of GPI structure. Interestingly, the mannan chain of the GM is the elongation of the mannan moiety of GPI structure. Interestingly, the mannan chain of the GM is composed of repeat units of 4 mannose residues with 3 α-1,2 and 1 α-1,6 linkage similar to the common composed of repeat units of 4 mannose residues with 3 α-1,2 and 1 α-1,6 linkage similar to the common structure of fungal GPI. However, one question remains open: does the LGM follow the GPI-pathway structure of fungal GPI. However, one question remains open: does the LGM follow the GPI-pathway of GPI-APs? of GPI-APs? Figure 1.1. ((A)) SchemeScheme ofof GlycosylphosphatidylinositolGlycosylphosphatidylinositol (GPI)(GPI) structure from GPI-anchoredGPI-anchored proteinsproteins (GPI-Aps)(GPI-Aps) inin A. fumigatusfumigatus;(; (B) SchemeScheme of thethe biosynthesisbiosynthesis of thethe GPIGPI precursorprecursor and itsits transfertransfer toto proteinprotein inin thethe ERER membranemembrane inin fungi.fungi. GlcNAcGlcNAc additionaddition toto PIPI andand de-N-acetylationde-N-acetylation of GlcNAc-PIGlcNAc-PI to GlcN-PI occuroccur atat the the cytoplasmic cytoplasmic face face of of the the ER ER membrane, membrane, and and further further additions additions to theto the GPI GPI occur occur on theon lumenalthe lumenal side ofside the of ER the membrane ER membrane [16] (reprinted [16] (reprint withed the with permission the permission from Genetics from SocietyGenetics of Society America). of (America).C) Scheme ( ofC) lipid Scheme remodeling of lipid of theremodeling GPI pathway of the in fungi. GPI pG1pathway and pG2: in phosphodiacylglycerol,fungi. pG1 and pG2: IPC:phosphodiacylglycerol, inositolphosphoceramide, IPC: inositolphosph M: mannose,oceramide, GN: glucosamine. M: mannose, GN: glucosamine. J. Fungi 2018, 4, 19 3 of 14 The GPI biosynthesis is a specific pathway that occurs at the endoplasmic reticulum (ER) membrane. Nine biochemical steps are required for the complete synthesis of the intermediate prior to being transferred to the target protein: transfer of N-acetylglucosamine (GlcNAc) onto a phosphatidylinositol (PI), GlcNAc deacetylation, inositol acylation, and the addition of 4 mannose and 3 phosphoethanolamine residues (Figure1)[ 17]. Apart from the phosphoethanolamine transfer onto the second mannose, all steps are essential to fungal growth [17]. This pathway is conserved